We have previously shown that phospholipase C-gamma (PLC-gamma) is activated in the presence of arachidnoic acid by tau, a neuronal cell-specific microtubule-associated protein. We have now purified another arachidonic acid-dependent PLC-gamma activating protein from non-neuronal tissue. Purified protein from bovine lung cytosol yielded multiple polypeptides ranging 70 - 130 kDa. Proteins eluted from each of the excised bands were able to activate PLC-gamma. Tryptic digestion and peptide sequencing identified the activator proteins as products of the AHNAK gene. The gene is known to encode an exceptionally large protein ( 700 kDa) with a very repetitive structure but no specific function has yet been assigned. The protein sequence deduced from human genomic sequence is composed of more than 30 of highly conserved 128-residue repetitive domains flanked by N- and C-terminal domains with unique sequences. The polypeptides isolated as PLC-gamma activators appeared to be splice variants or proteolytic fragements of AHNAK, which contained only several of the repetitive domains and lacked the unique ? and C-terminal domains. Two fragments of AHNAK, one containing four repeats and the other containing one repeat, were expressed as GST fusion protein. Both recombinant proteins activated PLC-gamma at concentrations as low as nanomolar in the presence of arachidonic acid. We showed that arachidonic acid promotes a physical interaction between AHNAK and PLC-gamma. As a consequence of the binding to AHNAK, the enzyme's apparent Km toward the substrate phosphatidylinositol 4,5-bisphosphate was reduced. These results suggest that arachidonic acid generated by phospholipase A2 can serves an an activator for PLC-gamma independently of tyrosine phosphorylation.